Aging, particularly of the skin, involves important intimate tissue biochemical disturbances which are manifested by macroscopic modifications, conventionally judged to be undesirable, and which continuously preoccupy both women and men.
Suntanning with natural solar UV, or artificially in beauty salons, is responsible for cutaneous aging well known to dermatologists under the name heliodermia (Dr. C. Musy-Preault, (1994) The Maladies of the Skin, Albin Michel ed., Paris).
Other components of our present way of life, such as physical and chemical aggression by pollution; the consumption of alcohol and tobacco, promote and aggravate the aging processes.
Moreover, in the course of private or professional life, the skin, first barrier of the organism against the outside world, is menaced in its integrity by numerous localized aggressions such as cuts, burns, inflammatory reactions. To correct these, the organism has developed a series of reactions, complex and overlapping each other: healing.
The cosmetic industry is continuously searching for new ingredients capable of countering the effects of aging in general and/or promoting cutaneous healing.
To do this, one of the possible approaches consists in promoting the tissue restructuring by neosynthesis of the different elements constituting the skin. Just as cement ensures the cohesion of bricks in a wall and gives it its solidity, the different types of collagens and other mucupolysaccharides, are the constituent elements of cutaneous tissue.
Promoting the synthesis and the incorporation of these molecules is surely necessary but not sufficient by itself. It is also necessary to prepare the terrain by giving it a good basis on which the mechanisms of healing can carry out lasting repairs. In the situations described above, this basis is the extracellular matrix, which is known as the basal layer when it is located at the interface of the epithelium and conjunctive tissue. Improvement or reconstruction of the extracellular matrix is important because it is now known that not only this structure plays xe2x80x9cthe role of framework stabilizing the physical structure of the tissuesxe2x80x9d but it also xe2x80x9cplays a role . . . in the regulation of the behavior of the cells which are in contact with itxe2x80x94influencing their development, their migration, their proliferization, their form and their functionsxe2x80x9d (Molecular Biology of the Cell, 3rd edition Medicine-Science, Flammarion, Paris, page 972).
There is accordingly a special interest in two of the principal constituents of this extracellular matrix: the collagens and the glycosaminoglycans (also known as GAGS).
In the framework of this patent, the effects of aging on the collagens and the glycosaminoglycans can be summarized as:
The decrease of synthesis of these molecules by fibroblasts, decrease due to the conjunction of two causes: on the one hand the quantity of renewal of these productive cells decreases with age and, on the other hand the quantity of molecules secreted by these cells also decreases.
When it is considered that collagen represents about 80% of the cutaneous proteins, it is easy to understand that the slightest decrease in its tissue concentration can have important consequences on the mechanical and physiological properties of the skin.
The glycosaminoglycans are capable of fixing large quantities of water. The decrease of their tissue concentration thus gives rise to cutaneous dehydration.
The appearance of structural modifications of the neo-synthesized molecules which leads to the reticulation of the fibers and hence their rigidification.
For collagen, the variations of the a chains modify the distribution of these different forms. For example, the proportion of type III collagen increases in the epidermis when the type IV collagen accumulates in the basal membrane. There have also been observed the appearance of reactions, enzymatic or not (of the Maillard reaction type) which create connections, called crossings, either between two fibers of collagen, or between the collagen itself and glucose molecules, thereby rigidifying the networks of collagen fibers.
Aging manifests itself in the glycosaminoglycans by the imperfect synthesis of their polysaccharide chains and by a decrease in their sulfation. More than with collagens, the radical forms of oxygen degrade the GAGs in an irreversible manner.
The skin thus loses its substance by the decrease in the quantity of its constituents, hardens by the loss of elasticity of the collagen fibers and by dehydration.
All this contributes to giving the aged skin its characteristic appearance: dryness, absence of flexibility, fineness, fragility, more or less numerous wrinkles that are more or less deep.
Healing itself requires, at least partially, similar needs because it is necessary to reconstruct and hence to produce a tissue mass; this implies locally, the increased synthesis of the different cutaneous constituents.
Thus, any product capable of inducing one or more processes increasing locally the synthesis of collagens and glycosaminoglycans, will permit obtaining the effect sought by all those who wish to reduce the cutaneous marks of aging as well as those wishing to improve healing, not only to quicken it but also for the aesthetics and quality of the result.
The invention which is the object of this patent application resides in the fact that we have developed a product which responds to the preceding criteria and of which we have demonstrated the efficacy, in vitro and in vivo, by sophisticated scientific tests.
It is known that the synthesis of collagen can be stimulated (in vitro), in cell cultures, by the C-terminal fragment of collagen I which constitutes the peptide Lys-Thr-S Thr-Lys-Ser (Katayama K. et al., Journal of Biological Chemistry (1993), 259:9941-9944.
Moreover, it is possible to increase the synthesis of the cutaneous glycosaminoglycans by vegetal extracts (for example, in the rat: Chithra P. et al., Journal of Ethnopharmacology (1998), 59:179-186).
Our patent application rests on the discovery, that when administered, alone or in combination with each other, by topical route in vivo, and hence by a method suitable for cosmetics, the peptides of the general formula R1-X-Thr-Thr-Lys-(AA)n-Y and their salts, wherein:
X representing a basic amino acid of D or L orientation (lysine, arginine, histidine, ornithine, citrulline, sarcosine, statine),
(AA)n representing a chain of n amino acids, natural or not, with n varying from 0 to 5,
R1 being H or a fatty acid chain of 2 to 22 carbons, hydroxylated or not, saturated or not, linear or branched, sulfurated or not, cyclic or not, or a biotin group, or a protective group of the urethane type used in peptide synthesis such as the groups benzyloxycarbonyl (Z), terbutyloxycarbonyl (tBoc), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc)
Y=OR2 or NR2R3 wherein R2 and/or R3 are a hydrogen atom or an aliphatic or aromatic chain of 1 to 22 carbons, hydroxylated or not, saturated or not, linear or branched, sulfurated or not, cyclic or not,
except peptides wherein R1=H and X=Lys and Y=OH and with n=0 or (AA)n=Ser when n=1,
are capable of increasing in a substantial way the concomitant synthesis of collagen and glycosiminoglycans and that this fact permits obtaining a synergetic effect because thus, the observed result is greater than could be hoped for from the addition of each of its effects.
Thus, the newly-formed collagen fibers overlay each other immediately in the trellis of the glycosiminoglycans of the basal layer newly synthesized; thereby accelerating the process of cutaneous regeneration as well as the mean level of tissue hydration.
The peptides preferably used in this way can be characterized in that n=1, R1 is a fatty acid chain of 2 to 22 carbons and Y is OH or NH2, and more precisely with X=lysine, (AA)n=serine, R1=the palmitoyl group and Y=OH.
The peptides which are the objects of this application can be obtained either by conventional chemical synthesis (in heterogenous phase or homogeneous phase), or by enzymatic synthesis (Kullman et al., J. Biol. Chem. 1980, 255, 8234) from constituent amino acids or their derivatives.
The small size of these peptides permits making them by industrial synthesis, at a good cost. Their demonstrated high activity permits the commercial use of them in a large number of cosmetic or dermopharmaceutical products that are financially favorable.
The peptide can be obtained also by fermentation of a strain of bacteria, modified or not by genetic engineering, to produce the sought sequences or their different fragments.
Finally, the peptide can be obtained by extraction of proteins of animal or vegetable origin, preferably vegetable, adapted to contain these sequences in their structure, followed by controlled hydrolysis, enzymatic or not, which frees the peptide fragments in question (of the sequence X-Thr-Thr-Lys-(AA) preferably Lys-Thr-Thr-Lys-Ser), of a mean size comprised between 300 and 2000 daltons, with the stipulation that the freed fragments correspond to the preceding peptide synthesis in plants which are adapted to contain these sequences within their structure. The managed hydrolysis permits freeing these peptide fragments.
To carry out the invention, it is possible but not necessary, either to extract the proteins in question first and then to hydrolyze them, or to carry out hydrolysis first on a raw extract and to purify the peptide fragments. There can also be used the hydrolysate without extracting from it the peptide fragments in question, by ensuring however the stopping of the enzynamic hydrolysis reaction timewise and determining the presence of the peptides in question by suitable analytic means (radioactivity tracing, immunofluorescence or immunoprecipitation with specific antibodies, etc.).
Other simpler or more complex processes, leading to less costly or more pure products, are easily envisagable by those skilled in the art knowing the extraction and purification procedures for proteins and peptides.